Bacterial composition and its use

ABSTRACT

The subject of the present invention is a bacterial composition having immunomodulation properties comprising at least one strain selected from the group consisting of  Lactobacillus acidophilus  PTA-4797,  Lactobacillus plantarum  PTA-4799,  Lactobacillus salivarius  PTA-4800,  Lactobacillus paracasei  PTA-4798,  Bifidobacterium bifidum  PTA-4801 and  Bifidobacterium lactis  PTA-4802. An other subject of the invention is an immunomodulation method comprising the step of using the at least one strain selected from the preceding group.

This application claims the priority benefit of U.S. application Ser.No. 11/594,543, filed Nov. 8, 2006, the disclosure of which isincorporated herein by reference, which claims the benefit of U.S.application Ser. No. 10/310,549, filed Dec. 5, 2002.

The subject of the present invention is a bacterial composition, animmunomodulation method and the use of this composition.Immunomodulation is the capacity to improve the global immune functionseither in healthy or in pathologic situations.

Among bacteria, some have a positive influence on the immune system ofthe intestinal medium, in particular lactic acid bacteria andbifidobacteria, and are termed “probiotic” bacteria or strains.

The expression probiotic bacteria or strains is understood to mean astrain which, when ingested live, exerts a beneficial effect on the hostby having an action on the balance of the intestinal flora. Theseprobiotic strains have the capacity to survive following passage acrossthe upper part of the digestive tube. They are non-pathogenic, non-toxicand exert a beneficial action on health through in one hand theirecological interactions with the resident flora of the digestive tractand in the other hand their ability to positively influence immunesystem, through their effects onto the GALT (gut associated immunetissue). According to the probiotic definition, those bacteria whengiven in sufficient numbers have the capacity to transit alive all alongthe gut. There they become part of the resident flora or the period ofadministration. The so called colonization (or transient colonization)allow the probiotic bacteria to exert beneficial effect, such asrepression of potentially pathogen micro-organisms on the flora andinteractions with the gut immune system.

The probiotic strains most widely used, in particular in dairy products,are mainly bacteria and yeasts, of the following genera Lactobacillusspp, Streptococcus spp, Enterococcus spp and Bifidobacterium spp andSacharomyces spp. Among the probiotic effects reported for thosebacteria, one can cite for example, improvement of lactose tolerance,prevention and or treatment of gastrointestinal and urogenitalinfections, reduction of some cancers, decrease of blood cholesterol.However, it should be highlighted that not all the individual strainsfrom the genera described above have those effects, but only somecarefully selected strains do.

Thus in order to satisfy the requirements for performant probioticstrains, it has become necessary to select strains or a mixture thereofwhich is efficient and which allow stimulation of the immune system(immunomodulation).

Accordingly, the problem which the present invention proposes to solveis to provide a bacterial composition having probiotic properties.

The aim of the present invention is to satisfy these requirements.

For this purpose, the present invention provides a bacterial compositionhaving immunomodulation properties comprising at least one strainselected from the group consisting of Lactobacillus acidophilusPTA-4797, Lactobacillus plantarum PTA-4799, Lactobacillus salivariusPTA-4800, Lactobacillus paracasei PTA-4798, Bifidobacterium bifidumPTA-4801 and Bifidobacterium lactis PTA-4802.

Another subject of the invention is an immunomodulation method.

The subject of the invention is also a food or pharmaceuticalcomposition comprising the bacterial compositions described above.

Another subject of the invention is also the use of this composition inthe preparation of a carrier administered to humans or to animals for atherapeutic or prophylactic purpose in the gastrointestinal system.

The composition of the invention has the advantage of providingunquestionable virtues which enrich the range of available strains.

Such a composition is particularly advantageous when it is administeredto humans or to animals for a therapeutic or prophylactic purpose in thegastrointestinal system, in particular in the reduction of inflammatoryand/or allergic reactions.

The advantage of the present invention is also to preserve all itsproperties when it is incorporated into a pharmaceutically acceptablecarrier or into a food product.

Other advantages and characteristics of the present invention willemerge more clearly on reading the description and the examples givenpurely by way of illustration and without limitation, which follow.

First of all, the subject of the invention is a bacterial compositionhaving immunomodulation properties comprising at least one strainselected from the group consisting of Lactobacillus acidophilusPTA-4797, Lactobacillus plantarum PTA-4799, Lactobacillus salivariusPTA-4800, Lactobacillus paracasei PTA-4798, Bifidobacterium bifidumPTA-4801 and Bifidobacterium lactis PTA-4802.

The Lactobacillus acidophilus used according to the invention is aGram-positive strain. Advantageously, it is a catalase-negative strain,with a homofermentative metabolism giving rise to the production oflactic acid.

The Lactobacillus acidophilus used according to the invention may alsoproduce a bacteriocin, lactucin, which is active against othermicroorganisms.

Preferably, it is a Lactobacillus acidophilus exhibiting good resistanceto pepsin, under acidic pH conditions (about at least 73% of thebacteria are still alive after 40 minutes of treatment).

More particularly, the Lactobacillus acidophilus used according to theinvention exhibits a very good resistance to pancreatin (at least 100%of the bacteria are still alive after 40 minutes of treatment).

Advantageously, the Lactobacillus acidophilus used according to theinvention exhibits good tolerance to bile salts.

Preferably, a Lactobacillus acidophilus described as being“hydrophobic”, that is to say which exhibits a high affinity forhydrophobic organic solvents, polar or not polar, such as for examplen-decane, chloroform, hexadecane or xylene, will be used.

The Lactobacillus acidophilus used according to the invention can inducethe production of cytokines. This detection of the induction ofcytokines was made by means of a test for in vitro stimulation ofisolated peripheral blood mononuclear cells (PBMC). Among the cytokinesinduced during this test, there may be mentioned interleukins 10 (IL10),γ-interferon (γ-IFN) and tumour necrosis factor α (TNFα). On the otherhand, the Lactobacillus acidophilus used according to the inventioninduces little or no secretion of interleukins 12 (IL12) using this sametest.

The Lactobacillus acidophilus used according to the invention isLactobacillus acidophilus PTA-4797. This Lactobacillus acidophilusstrain was deposited according to the Treaty of Budapest at the AmericanType Culture Collection where it is recorded under the deposit numberPTA-4797.

One embodiment of the composition according to the invention is abacterial composition comprising Lactobacillus acidophilus PTA-4797.

The composition made of probiotic bacteria according to the inventionmay also comprise at least one Lactobacillus plantarum strain.

The Lactobacillus plantarum strain used according the invention ispreferably a Gram-positive strain. Advantageously, it is acatalase-negative strain, with a homofermentative metabolism giving riseto the production of lactic acid.

Preferably, it is a Lactobacillus plantarum which is resistant to pepsinunder acidic pH conditions (about at least 95% of the bacteria are stillalive after 40 minutes of treatment).

More particularly, the Lactobacillus plantarum used according to theinvention exhibits good resistance to pancreatin (about at least 79% ofthe bacteria are still alive after 40 minutes of treatment).

Advantageously, the Lactobacillus plantarum used according to theinvention exhibits good resistance to bile salts.

The Lactobacillus plantarum used according to the invention can inducethe production of cytokines. This detection of the induction ofcytokines was made by means of a test for in vitro stimulation ofisolated peripheral blood mononuclear cells (PBMC). Among the cytokinesinduced during this test, there may be mentioned interleukins 10 (IL10),γ-interferon (γ-IFN) and tumour necrosis factor α (TNFα). On the otherhand, the Lactobacillus plantarum used according to the inventioninduces little or no secretion of interleukins 12 (IL12) using this sametest.

The Lactobacillus plantarum used according to the invention isLactobacillus plantarum PTA-4799. This Lactobacillus plantarum strainwas deposited according to the Treaty of Budapest at the American TypeCulture Collection where it is recorded under the deposit numberPTA-4799.

One embodiment of the composition according to the invention is abacterial composition comprising Lactobacillus plantarum PTA-4799.

The composition made of probiotic bacteria according to the inventionmay also comprise at least one Lactobacillus salivarius strain.

The Lactobacillus salivarius strain used according to the invention is aGram-positive strain. Advantageously, it is a catalase-negative strain,with a homofermentative metabolism giving rise to the production oflactic acid.

More particularly, the Lactobacillus salivarius used according to theinvention exhibits good resistance to pancreatin (at least 100% of thebacteria are still alive after 40 minutes of treatment).

The Lactobacillus salivarius used according to the invention can inducethe secretion of cytokines. This detection of the induction of cytokineswas made by means of a test for in vitro stimulation of isolatedperipheral blood mononuclear cells (PBMC). Among the cytokines inducedduring this test, there may be mentioned interleukins 10 (IL10) andtumour necrosis factor α (TNFα). On the other hand, the Lactobacillussalivarius used according to the invention induces little or nosecretion of interleukin 12 (IL12) and γ-interferon (γ-IFN) using thissame test.

The Lactobacillus salivaris used according to the invention isLactobacillus salivarius PTA-4800. This Lactobacillus salivarius strainwas deposited according to the Treaty of Budapest at the American TypeCulture Collection where it is recorded under the deposit numberPTA-4800.

One embodiment of the composition according to the invention is abacterial composition comprising Lactobacillus salivarius PTA-4800.

The composition made of probiotic bacteria according to the inventionmay also comprise at least one Lactobacillus paracasei strain.

The Lactobacillus paracasei used according to the invention is aGram-positive strain. Advantageously, it is a catalase-negative strain,with a homofermentative metabolism giving rise to the production oflactic acid.

Preferably, it is a Lactobacillus paracasei exhibiting poor resistanceto pepsin, under acidic pH conditions (about at least 17.5% of thebacteria are still alive after 40 minutes of treatment).

More particularly, the Lactobacillus paracasei used according to theinvention exhibits a very good resistance to pancreatin (about at least100% of the bacteria are still alive after 40 minutes of treatment).

Advantageously, the Lactobacillus paracasei used according to theinvention exhibits good tolerance to bile salts.

The Lactobacillus paracasei used according to the invention can inducethe production of cytokines. This detection of the induction ofcytokines was made by means of a test for in vitro stimulation ofisolated peripheral blood mononuclear cells (PBMC). Among the cytokinesinduced during this test, there may be mentioned interleukins 10 (IL10),γ-interferon (γ-IFN) and tumour necrosis factor α (TNFα). On the otherhand, the Lactobacillus paracasei used according to the inventioninduces little or no secretion of interleukins 12 (IL12) using this sametest.

The Lactobacillus paracasei used according to the invention isLactobacillus paracasei PTA-4798. This Lactobacillus paracasei strainwas deposited according to the Treaty of Budapest at the American TypeCulture Collection where it is recorded under the deposit numberPTA-4798.

One embodiment of the composition according to the invention is abacterial composition comprising Lactobacillus paracasei PTA-4798.

The composition made of probiotic bacteria according to the inventionmay also comprises at least one Bifidobacterium bifidum strain.

The Bifidobacterium bifidum strain used according to the invention is aGram-positive strain. Advantageously, it is a catalase-negative strain,with a heterofermentative metabolism giving rise to the production oflactic acid and acetic acid.

The Bifidobacterium bifidum used according to the invention isBifidobacterium bifidum PTA-4801. This Bifidobacterium bifidum strainwas deposited according to the Treaty of Budapest at the American TypeCulture Collection where it is recorded under the deposit number ATCCPTA-4801.

One embodiment of the composition according to the invention is abacterial composition comprising Bifidobacterium bifidum PTA-4801.

The composition made of probiotic bacteria according to the inventionmay also comprises at least one Bifidobacterium lactis strain.

The Bifidobacterium lactis strain used according to the invention is aGram-positive strain. Advantageously, it is a catalase-negative strain,with a heterofermentative metabolism giving rise to the production oflactic acid and acetic acid.

The Bifidobacterium lactis used according to the invention isBifidobacterium lactis PTA-4802. This Bifidobacterium lactis strain wasdeposited according to the Treaty of Budapest at the American TypeCulture Collection where it is recorded under the deposit number ATCCPTA-4801.

One embodiment of the composition according to the invention is abacterial composition comprising Bifidobacterium lactis PTA-4801.

Another embodiment of the composition according to the invention is abacterial composition comprising at least one strain selected from thegroup consisting of Lactobacillus acidophilus PTA-4797, Lactobacillusplantarum PTA-4799, and Bifidobacterium lactis PTA-4802.

More particularly, the composition according to the invention maycomprise at least 2 strains selected from the group consisting ofLactobacillus acidophilus PTA-4797, Lactobacillus plantarum PTA-4799,Lactobacillus salivarius PTA-4800, Lactobacillus paracasei PTA-4798,Bifidobacterium bifidum PTA-4801 and Bifidobacterium lactis PTA-4802.

More particularly, the composition according to the invention maycomprise 3 strains selected from the group consisting of Lactobacillusacidophilus PTA-4797, Lactobacillus plantarum PTA-4799, Lactobacillussalivarius PTA-4800, Lactobacillus paracasei PTA-4798, Bifidobacteriumbifidum PTA-4801 and Bifidobacterium lactis PTA-4802.

Preferably, the composition according to the invention may comprise ablend of Lactobacillus acidophilus PTA-4797, Lactobacillus plantarumPTA-4799, Lactobacillus salivarius PTA-4800, Lactobacillus paracaseiPTA-4798, Bifidobacterium lactis PTA-4802 and Bifidobacterium bifidumPTA-4801.

The composition according to the invention may be used in the form of abacterial suspension, before or after freezing, or of a freeze-driedpowder. Indeed, regardless of the form used, the composition may befrozen.

The relative proportion of each bacterium in the composition can vary inlarge limit for example from 1/99 to 99/1 in the case there is at least2 strains.

The composition according to the invention may comprise from 10⁶ to 10¹¹CFU of bacteria/g of composition, and more particularly from 10⁸ to 10¹¹CFU of bacteria/g of composition. The term CFU means “colony formingunits”. The expression gram of composition is understood to mean thefood product or pharmaceutical preparation, and preferably 10⁹ to 10¹¹CFU/g if in a freeze-dried form.

The composition according to the invention is useful for the treatment,the primary prevention or the recurrence, and also the prevention and/orthe reduction of inflammatory bowel disease.

The bacterial composition according to the invention is also useful formaintaining the homeostasis of the immune system.

The bacterial composition according to the invention is also useful forthe prevention and/or the reduction of allergenicity.

The bacterial composition according to the invention is also useful asimmunoadjuvant.

An other subject of the invention is also an immunomodulation methodcomprising the step of using at least one strain selected from the groupconsisting of Lactobacillus acidophilus PTA-4797, Lactobacillusplantarum PTA-4799, Lactobacillus salivarius PTA-4800, Lactobacillusparacasei PTA-4798, Bifidobacterium bifidum PTA-4801 and Bifidobacteriumlactis PTA-4802.

The composition according to the invention may be used in the form of afood product or pharmaceutical preparation.

The expression pharmaceutical preparation is understood to mean forexample a preparation in the form of capsule or tablet.

The subject of the invention is also a food, dietary supplement orpharmaceutical composition comprising the bacterial compositiondescribed above.

Preferably, the food composition comprising the composition according tothe invention is a food supplements, a beverage product or a milk basepowder.

More particularly, the food composition comprising the compositionaccording to the invention is a dairy product.

More preferably the food composition comprising the compositionaccording to the invention is an infant formula.

The food or pharmaceutical composition according to the invention mayhave immunomodulation properties.

Another subject of the invention is also the use of this composition inthe preparation of a carrier administered to humans or to animals for atherapeutic or prophylactic purpose in the gastrointestinal system.

The invention provides also a pharmaceutical composition comprising thebacterial composition described above useful for the prevention ofinflamatory bowel disease.

The invention provides also a pharmaceutical composition comprising thebacterial composition described above useful for immunomodulation.

The FIGS. 1, 3 and 5 are schedules of injection.

The FIGS. 2, 4 and 6 are Wallace and Ameho scores, and weight of loss.

Concrete but nonlimiting examples of the invention will now bedescribed.

EXAMPLES

1/ PBMC Test:

PBMC (Peripheral Blood Mononuclear Cells) are prepared by centrifugationfrom human blood, derived from known donors and is further purified on aFicoli gradient. Cells are harvested, washed, (red blood cells removed)and counted.

Bacteria are prepared according to standard conditions and counted byplating on agarose medium according to proper dilutions (10⁻⁷, 10⁻⁸,10⁻⁹ in Ringer solution).

Cells are washed 3 times and suspended (et non dissolved) in PBS buffer.A verifier par Dominique

PBMC cells are stimulated for 48 hours with the bacteria (allow negativecontrol with Phosphate Buffer Saline solution (PBS)) under appropriateconditions of CO₂. Then the supernatant containing the cytokines isfrozen at −20° C.

Cytokines expression levels are determined by ELISA tests (<<Enzymelinked immuno sorbent assay>>). ELISA plates are coated withanti-cytokine antibody (overnight procedure) and the antibody is blockedwith a surfactant, the tween 80.

A proper standard is prepared for known concentrations of cytokineswhich will cover the detection range of 15.62 till 2000 pg/ml (incubateovernight). Perform the anti-cytokine detection and quantify withstreptavidin reaction on substrate (10 mg dABTS/10 ml of citric acidbuffer 0.1 M, pH4.35/20 μl H2O2).

Cytokines are either pro-inflammatory/Th1 (TNFα, IFNγ, IL12) oranti-inflammatory (IL10).

Strains IL-10 IL-12 IFN-γ TNF-α L. acidophilus PTA-4797 309 1970 27591L. salivarius PTA-4800 6881 31 1148 23509 L. paracasei PTA-4798 300 31921 14046The values are pg/ml.

2/ Preparation of a Composition made of Lactobacillus acidophillusPTA-4797, Lactobacillus plantarum PTA-4799 and Bifidobacterium lactisPTA-4802:

A blend of 3 strains is prepared containing Lactobacillus acidophilusPTA-4797, Lactobacillus plantarum PTA-4799, et Bifidobacterium lactisPTA 4802. This blend is named LAB.

To show in vivo, the immuno modulation capacity of this blend accordingto the invention, animal test were performed aimed at decreasing thechemically induced gut inflammation of mice. Several models were set-upto mimic human inflammatory bowel diseases.

In the first model, injections of 1 mg/mice of TNBS (trinitrobenzenesulfonic acid, which is a colitis inducing agent) on days 0, 7 and 14,and 2 mg/mice of TNBS on day 20 (double dose) will evoke a chroniccolitis. The schedule of injection is drawn on FIG. 1. The intake ofbacteria (LAB) on days −4 to −1, 6, 9, and 19 will show macroscopic andhistological effects, which are scored on day 22, according to standardscores (Wallace and Ameho respectively).

The results obtained (see FIG. 2) clearly showed an improvement of thecolitis symptoms evidenced by

reduction of the increase of the submucosa

reduction of the inflammation

less weight loss

A second model of chronic colitis was also used (based on Camoglio etal., Eur J Immunol 2000), 2 mg/mice of TNBS injected by intra-rectalroute on days 0, 7, and 14 induce a serious chronic colitis. Theschedule of injection is drawn on FIG. 3. The intake of bacteria (LAB)on days −5 to −1, on days 5 and 12 (FIG. 3) will show macroscopic andhistological effects, which are scored on day 16, according to standardscores (Wallace and Ameho respectively).

The results obtained clearly showed an improvement of the colitissymptoms evidenced (see FIG. 4), by

absence of necrotic lesions

reduction of the increase of the submucosa

reduction of the inflammation

A third acute model of mice colitis was also set up. 100 mg/kg mice ofTNBS is injected on day 0. This will evoke an acute colitis. Theschedule of injection is drawn on FIG. 5. The intake of Bifidobacteriumlactis PTA-4802 or Lactobacillus plantarum PTA-4799 (10⁷ bacteria/mouse)on days −5 to 0 show macroscopic and histological effects which arescored on day 2.

Clear macroscopic improvement of the colitis symptoms were evidenced forstrains Bifidobacterium lactis PTA-4802 and Lactobacillus plantarumPTA-4799 (FIG. 6).

In general all the TNBS-induced colitis models in mice show that feedingLAB to these mice significantly reduced the level of intestinaltranslocation of indigenous bacteria from the intestinal flora intomesenteric lymph nodes and spleen after the induction of colitis. Inaddition, no translocation of probiotic bacterial strains according tothe invention was observed.

3/ Resistance to Pepsin

The aim of this test is to evaluate the resistance of bacteria to thepassage of the gastric barrier by determining the number of bacteriathat survive after cultivation in the presence of a pepsin solution inacid environment. In short, 100 μl of a stock culture frozen at −80° C.is inoculated in 10 ml of MRS (Man Rogosa Sharp) culture medium andincubated overnight at 30° C. or 37° C. Then cells are washed 3 times inPBS at pH7 and the pellet is suspended in 1 ml PBS buffered andinoculated in 200 μl aliquots in 4 tubes with 1 ml of filtered pepsinsolution at pH 2 supplemented with 300 μl of NaCl (0.5%). Cell count isperformed on 100 μl aliquots taken from the tubes incubated at T0 (timeof inoculation), T0+5 min; T020 min; T0+40 min; T0+60 min and dilutionsare plated on MRS (100 μl) of 10⁻⁵ 10⁻⁶ et 10⁻⁷ (consecutive 10 folddilutions made in 1 ml Ringer solution). The percentage survivingbacteria is calculated for each incubation time from the reading of thedilution series.

All results displayed in the tables are the result of triplicateexperiments

Strains 5 min 20 min 40 min 60 min L. acidophilus PTA-4797 91 74 73 55L. plantarum PTA-4799 100 100 95 42 L. salivarius PTA-4800 100 66 23 7L. paracasei PTA-4798 100 17.5 17.5 1.5Results as % of survival compared to T=0.

4/ Resistance to Pancreatin

The aim of this test is to evaluate the resistance of bacteria to thepassage of the intestinal transit by determining the number of bacteriathat survive after cultivation in the presence of a pancreatin solutionin basic environment. In short, 100 μl of a stock culture frozen at −80°C. is inoculated in 10 ml of MRS (Man Rogosa Sharp) culture medium andincubated overnight at 30° C. or 37° C. Cells are washed 3 times in PBSat pH7 and the pellet is suspended in 1 ml PBS buffered and inoculatedin 200 μl aliquots in 4 tubes with 1 ml of filtered pancreatin solutionat pH 8 supplemented with 300 μl of NaCl (0.5%). Cell count is performedon 100 μl aliquots taken from the tubes incubated at T0 (time ofinoculation), T0+5 min; T0+20 min; T0+40 min; T0+60 min; T0+120 min anddilutions are plated on MRS (100 μl) of 10⁻⁵, 10⁻⁶, 10⁻⁷ (consecutive 10fold dilutions made in 1 ml Ringer solution). The percentage survivingbacteria is calculated for each incubation time from the reading of thedilution series. All results displayed in the tables are the result oftriplicate experiments.

Strains 5 min 20 min 40 min 60 min 120 min L. acidophilus 83 100 100 170170 PTA-4797 L. salivarius 100 115 152 158 155 PTA-4800 L. plantarum 8672 79 84 87 PTA-4799 L. paracasei 65 100 100 100 100 PTA-4798Results as % of survival compare to T=0.

5/ Resistance to Bile Salts

The experimental is procedure is as follows. 100 μl culture of a stockfrozen at −80° C. is inoculated in 10 ml de milieu de culture MRS (ManRogosa Sharp) and incubated overnight at 30° C. or 37° C. The opticaldensity (OD) is measured and a dilution is prepared (OD=0.05 to 0.1) intwo bottles. One of the culture bottles is supplemented with a 0.3%solution of bile salts (750 μl of a filtered 12% solution) and incubatedat the correct temperature. The OD is regularly measured until a OD of0.3 is reached in the control bottle without bile salts. At that pointthe OD of the supplemented bottle is measured and one starts to measurethe time needed to obtain the same OD of 0.3. The resistance of thestrain is therefore evaluated as the delay in growth time.

Results are coded as sensitive (0; more than 60 minutes), tolerant (1;between 15 and 60 minutes) or resistant (2; less than 15 minutes).

All results displayed in the tables are the result triplicateexperiments

Strains Resistance L. acidophilus PTA-4797 1 L. plantarum PTA-4799 2 L.paracasei PTA-4798 2

6/ Hydrophobicity and Affinity for Organic Solvents

The MATS test as described by Rosdenberg et al., 1980 (FEMS Microbiol.Letters 9; 29-33) has been used. In short the test measures (grownovernight under normal in vitro growth conditions and washed twice inPBS) the hydrophobicity and polarity of the bacterial cell wall, basedon the repartition (measured by optical density) of the bacteria intotwo non mixable liquid aqueous phases and five respective solvents.Growth medium is MRS (Man Rogosa Sharp) and the aqueous phase is abuffer as PBS. The solvents were decane, hexadecane, ethyl acetate,chloroform and xylene.

The affinity for chloroform, acid solvent, reflects the electron donornature of the bacterium (basic reaction)

The affinity for Ethyl Acetate, basic solvent, reflects the electronacceptor nature of the bacterium (acid reaction)

The affinity for apolar solvents (decane, hexadecane, xylene) reflectsthe hydrophobic character of the bacterium.

Possibly a high hydrophobicity is related to the presence ofglycoproteins on the cell wall surface, while a low hydrophobicity couldbe linked to the presence of polysaccharides on the cell wall surface.% D′HYDROPHOBICITY=(1−A/A0)×100 with

AO: Original OD at 540 nm (set more or less at OD=0.6 in PBS)

A: Optical density of ageous phase (for 1 ml) after vortexing andstabilisation period

Ethyl Strains Decane Hexadecane acetate Chloroform Xylene L. acidophilus81.9 60.4 44.2 90.3 90.9 PTA-4797 L. salivarius 23.02 38.17 2.25 79.2851.18 PTA-4800 L. paracasei 36.5 37.4 28.8 45.2 30.6 PTA-4798 L.plantarum 25.3 25.6 28.7 28.8 21.9 PTA-4799

1. A method for modulating the immune system of a human or animal inneed thereof, said method comprising the step of administering to saidhuman or animal in need thereof an effective amount of a bacterialcomposition comprising Lactobacillus paracasei PTA-4798.
 2. The methodaccording to claim 1, wherein said method is a method for stimulatingthe immune system of a human or an animal in need thereof.
 3. The methodaccording to claim 1, wherein said bacterial composition furthercomprises at least one additional strain selected from the groupconsisting of Lactobacillus acidophilus PTA-4797, Lactobacillusplantarum PTA-4799, Bifidobacterium bifidum PTA-4801.
 4. The methodaccording to claim 1, wherein said bacterial composition furthercomprises at least two additional strains selected from the groupconsisting of Lactobacillus acidophilus PTA-4797, Lactobacillusplantarum PTA-4799, Bifidobacterium bifidum PTA-4801.
 5. The methodaccording to claim 1, wherein said bacterial composition comprises ablend of Lacrobacillus paracasei PTA-4798, Lactobacillus acidophilusPTA-4797, Lactobacillus plantarum PTA-4799, Bifidobacterium bifidumPTA-4801.
 6. The method according to claim 1, wherein said bacterialcomposition comprises from 10⁶ to 10¹¹ CFU of bacteria/g of composition.7. The method according to claim 1, wherein said bacterial compositioncomprises from 10⁸ to 10¹¹ CFU of bacteria/g of composition.